Production of cheese and cultured dairy products has long relied on the fermentation of milk by lactic acid-producing bacteria including species of Lactococcus. Since efficient fermentations are dependent on the growth and activity of these bacteria, great care is exercised to prepare starter cultures that are highly active and uncontaminated with undesirable microorganisms or bacteriophages. However, the fermentation process itself is nonaseptic, occurring in open vats with a nonsterile medium, e.g., pasteurized milk. It is, therefore, highly susceptible to contamination with bacteriophages. For the majority of strains of lactic acid bacteria employed in commercial dairy fermentations, lytic bacteriophages capable of halting growth and acid production can appear within one to two days after introducing the bacterial culture into the dairy plant.
Milk fermentations historically have relied on starter cultures composed of undefined mixtures of lactic acid bacteria propagated without knowledge of, or protection from, bacteriophages. Natural bacteriophage contamination in these cultures established an equilibrium of evolving bacteriophages and bacteriophage-resistant variants. These cultures were highly variable in day-to-day levels of acid production, but remained moderately active and could be used continuously in small fermentation factories. Over the past 20 years, starter culture failures due to bacteriophage infection have become prevalent throughout the dairy industry. Increasing demand for cultured milk products in recent years has necessitated increases in both production capacity and process efficiency such that larger volumes of milk are processed, cheese vats are filled repeatedly within a single day, and total processing time is shortened. This modernization of the industry concurrently increased the probability of bacteriophage contamination and further dictated the use of defined mixtures of lactic acid bacteria capable of uniform and rapid rates of acid production. With the selection of highly fermentative lactic acid bacteria and their propagation under aseptic conditions, i.e., in the absence of bacteriophages, the majority of cultures now used by the industry are susceptible to bacteriophage attack upon introduction into the cheese factory.
To cope with bacteriophage problems, a number of methods have been developed to minimize bacteriophage action during various fermentation processes, particularly dairy fermentations.
Bacteriophage resistance in various lactic acid bacteria is recognized generally as a plasmid-encoded phenomenon. For example, U.S. Pat. No. 4,732,859 to Hershberger et al. relates to a method of protecting various genera of bacteria from naturally occurring bacteriophage by providing host bacterial cells with a restriction system that digests HhaII site-containing foreign DNA, found in most naturally occurring phages. As bacteriophage DNA enters a host cell, the HhaII restriction endonuclease digests the DNA at HhaII sites and renders the bacteriophage non-functional and harmless. In more general terms, the invention involves transforming a bacterium with a recombinant DNA cloning vector which comprises a replicon that is functional in the bacterium, and a gene that expresses a functional protein (i.e., a restriction endonuclease which confers restriction activity to the bacterium).
U.S. Pat. Nos. 4,918,014 and 4,874,616, both to Vedamuthu, are directed to a method of imparting bacteriophage resistance to bacteriophage sensitive strands of Streptococcus group N, whereby a plasmid encoding the production of a mucoid substance is transferred via a plasmid into a bacteriophage sensitive strain. The lactic Streptococci, or "Lactococci, " are said to be rendered bacteriophage resistant, and purportedly more stable for use in milk fermentation.
Bacteriophage resistance in Lactococci also has been found generally to be a plasmid-encoded phenomenon. Plasmids have been described which direct resistance via inhibition of adsorption, restriction and modification ("R/M"), and by aborting the bacteriophage infection ("Hsp"). Strategies that will be useful for the construction of bacteriophage-insensitive strains include the introduction of one or more resistance mechanisms within a single host, or introducing a single plasmid containing more than one resistance mechanism. For recent reviews, see Klaenhammer, T. R., FEMS Microbiol. Rev. 46:313-325 (1987) and Sanders, Biochimie 70:411-422 (1988). A prerequisite for the success of these strategies is that the resistance mechanisms should work in combination to prevent bacteriophage proliferation. The insensitivity of any constructed, or natural, isolate is probably a function of time, amount of use, and environmental conditions (Klaenhammer, 1984, Lawrence and Thomas, 1979). The presence of single plasmids encoding multiple resistances, or combinations of plasmids within a single strain, can also confer prolonged resistance phenotypes upon lactococcal strains. Inevitably, however, defense mechanisms used for long periods have succumbed to an evolving bacteriophage population. This has occurred, for example, in the case of pTR2030 transconjugants of the industrial strain of L. lactis, NCK202. pTR2030 is a conjugative plasmid which confers resistance to bacteriophage via Hsp and R/M. Bacteriophages recovered from the industry after prolonged use of NCK202 have overcome either one or both of these mechanisms.
The search for novel resistance determinants to add to the growing arsenal of available, independent genotypes has continued. In particular, bacteriophage defense mechanisms active against those that are insensitive to Hsp would provide a valuable adjunct to existing resistance genotypes in bacteria, particularly lactic acid bacteria. Most avenues of bacteriophage replication could be blocked to minimize the adaptation and proliferation of new bacteriophages, which in turn would increase the longevity of fermentation bacterial cultures.